Recombinant AAV vectors are promising candidates for gene therapy of several human genetic disorders. Despite early successes in clinical trials, our understanding of the mechanisms underlying AAV host and tissue tropism is incomplete. During the previous funding period, we made significant progress in mapping novel AAV-glycan receptor interactions and engineering a new class of liver-detargeted AAV vectors. In addition, we optimized structural tools, in vitro cellular assays and in vivo animal studies pertinent to the characterization of AAV vectors. Although this information provided further insight into the biology of certain AAV serotypes, significant gaps exist in our understanding of numerous other AAV strains currently being tested in clinical studies. In the current proposal, we have assembled a team of experienced investigators in the fields of structural virology, glycobiology, vascular biology and AAV vectors to dissect the biology of several naturally occurring as well as new, lab-derived AAV mutants. Specifically, we will map the structural determinants of AAV-glycan interactions using a battery of molecular modeling, computational ligand docking, glycan array and molecular cloning tools. We then propose to reengineer glycan binding footprints on different AAV serotypes to generate novel mutants with altered tissue tropism. Comprehensive studies investigating the role of different tissue glycans as well as integrin co-receptors on the tropism, biodistribution, pharmacokinetics, and transvascular transport of AAV vectors using mouse models will also be undertaken. The long term goal of the proposed studies is to obtain a thorough understanding of AAV biology in different hosts. If successful, the current proposal could provide significant new insight into the influence of glycans and integrins on AAV tissue tropism. In addition to providing a roadmap for structure-driven design of improved AAV vectors, the proposed studies could guide the selection of appropriate AAV strains for further clinical development.